Light responses of rod and cone photoreceptors are encoded by the release of glutamate-filled vesicles at photoreceptor synapses. Synaptic transmission at the first synapse in the retina thus fundamentally shapes visual perception and damage to photoreceptor synapses by protein mutation or diseases such as macular degeneration and ischemia causes vision loss. To understand the consequences of damage to these synapses and how to restore vision by therapeutic means requires a thorough understanding of their normal operation. Release from photoreceptors involves a plate-like protein structure known as the synaptic ribbon. Unlike most central nervous system (CNS) synapses that release only one or two synaptic vesicles at a time, ribbon synapses in photoreceptors and other sensory neurons are specialized for continuous release. In addition to the ribbon itself, the specialized capabilities of ribbon synapses are also determined by the use of certain proteins that differ from those at more conventional synapses. Rod and cone photoreceptors differ further from both conventional and other ribbon synapses in their use of an exocytotic Ca2+ sensor with unusual Ca2+ dependence. At most synapses, synaptic vesicle release rate rises with the 5th power of [Ca2+]i but release from photoreceptors has a weaker 1-3rd order Ca2+-dependence. The identity of the atypical Ca2+ sensor that regulates vesicle release from photoreceptors is a major unresolved question about the mechanisms of release at the first synapse in the retina. Isoforms of the protein synaptotagmin (Syt) serve as the exocytotic Ca2+ sensors in most neurons. Our first aim is to identify the Ca2+ sensor controlling release from photoreceptors by testing mice in which specific Syt proteins have been selectively deleted from rods or cones. Our second aim is to confirm that the exocytotic Ca2+ sensors in mouse rod and cone synapses retain the unusually low Ca2+ cooperativity seen in lower vertebrates. In Aim 3, we propose to characterize how the Ca2+-dependence of release rate is shaped by different combinations of Syt, Complexin, and SNARE proteins that reproduce components of the rapid release machinery at different conventional and ribbon synapses, using unique in vitro approaches that can probe single fusion pores with sub-ms time resolution. Together, these experiments will reveal the mechanisms responsible for the atypical Ca2+-dependence of neurotransmission at the critical first synapse in vision and allow us to understand how the expression of particular proteins shapes the properties of release to meet specific signaling needs at different CNS synapses.